Fig 2: Cell proliferation and cell cycle analysis for the combination of CDK4 inhibitor and ATG5 or BECN1 knockdown. (A) Cell proliferation analysis under the conditions as described for Fig. 4B. Cell viability just before treatment with siRNA was 100%, and the vertical axis corresponds to the absorbance ratio. The x-axis indicates the type of treatment. The y-axis indicates the proportion of cell proliferation. Values shown are mean ± SD (n=3). (A) shows the data for the cell lines MDA-MB435S, BT474, MCF7, and KMST-6. Data for the other cell lines are shown in Table V. 1, DMSO + siControl; 2, DMSO + siATG5; 3, DMSO + siBECN1; 4, CDK4i + siControl; 5, CDK4i + siATG5; 6, CDK4i + siBECN1. (B) Cell cycle analysis was performed under the conditions as described for (A). The x-axis indicates the phase of the cell cycle. The y-axis indicates the proportion of the cell population. Values shown are mean ± SD (n=3). (B) shows the data for the cell lines MDA-MB435S, BT474, MCF7, and KMST-6. Data for the other cell lines are shown in Table VI. Differences in sub-G1 phase between DMSO + siControl and CDK4 inhibitor + siControl, between DMSO + siATG5 and CDK4 inhibitor + siATG5, and between DMSO + siBECN1 and CDK4 inhibitor + siBECN1 were analyzed using t-test. Lanes: 1, DMSO + siControl; 2, DMSO + siATG5; 3, DMSO + siBECN1; 4, CDK4i + siControl; 5, CDK4i + siATG5; 6, CDK4i + siBECN1. (C) Images of flow cytometry under the conditions as shown in (B). Lanes: 1, DMSO + siControl; 2, DMSO + siATG5; 3, DMSO + siBECN1; 4, CDK4i + siControl; 5, CDK4i + siATG5; 6, CDK4i + siBECN1. CDK4i, CDK4 inhibitor.

Fig 3: Flubendazole elicits anti-cancer effects via targeting EVA1A-modulated autophagy and apoptosis in TNBC cells. (A) MDA-MB-231 and MDA-MB-468 cells were transfected with negative-control or EVA1A siRNA, respectively. After treatment with or without flubendazole (0.5 µM) for 24 h, cell viability was measured by MTT assay. (B) MDA-MB-231 and MDA-MB-468 cells were transfected with negative-control or EVA1A siRNA, respectively. After treatment with or without flubendazole (0.5 M) for 2 weeks. Representative images and quantification of colonies were shown. (C) MDA-MB-231 and MDA-MB-468 cells were transfected with negative-control or EVA1A siRNA, followed by treatment with or without flubendazole (0.5 µM) for 24 h. Then, the expression levels of EVA1A, p62, LC3, Bax and Bcl-2 were determined by immunoblotting analysis. ß-actin was measured as a loading control. (D-E) MDA-MB-231 and MDA-MB-468 cells were transfected with negative-control or EVA1A siRNA, followed by treatment with or without flubendazole (0.5 µM) for 24 h. Representative images with quantification of LC3 intensity were shown. Scale bar, 10 µm. (F) MDA-MB-231 and MDA-MB-468 cells were co-transfected with EVA1A siRNA and Flag-EVA1A or vehicle control respectively for 24 h, followed by treatment with or without flubendazole (0.5 µM) for 24 h. Immunoblotting analysis of ATG5, EVA1A and LC3 expression. ß-actin was measured as a loading control. Quantification of immunoblotting were shown. (G-H) MDA-MB-231 and MDA-MB-468 cells were co-transfected with EVA1A siRNA and Flag-EVA1A or vehicle control respectively for 24 h, followed by treatment with or without flubendazole (0.5 µM) for 24 h. Representative images with quantification of LC3 intensity were shown. Scale bar, 10 µm. (I) MDA-MB-231 and MDA-MB-468 cells were co-transfected with EVA1A siRNA and Flag-EVA1A or vehicle control respectively for 24 h, followed by treatment with or without flubendazole (0.5 µM) for 24 h, cell viability was measured by MTT assay. (J) MDA-MB-231 and MDA-MB-468 cells were co-transfected with EVA1A siRNA and Flag-EVA1A or vehicle control respectively for 24 h. After treatment with or without flubendazole (0.5 µM) for 2 weeks. Representative images and quantification of colonies were shown. Data are expressed as mean ± SEM. All data were representative of at least three independent experiments. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Statistical significance compared with respective control groups.

Fig 4: Autophagy facilitates long-term (24 h) expression of VCAM-1 in HUVECs. (A,B) HUVECs were pretreated with or without 5 mmol/L 3-MA (A) or 100 nmol/L Bafilomycin A (BAF) (B) for 30 min followed by 10 ng/mL IL-1ß or 10 ng/mL TNFa for 24 h. VCAM-1 and ICAM-1 were analyzed by western blotting. Upper panel shows a representative blot and the lower panel the quantitative analysis. Each bar denotes mean ± SD of three independent experiments. *Indicates P < 0.05. (C) HUVECs were transfected with Atg5 siRNA or control siRNA for 72 h. Atg5 was analyzed by western blotting. (D) HUVECs were transfected with siRNA for 72 h followed by 10 ng/mL IL-1ß or 10 ng/mL TNFa for 24 h. VCAM-1, ICAM-1 and Atg5 were analyzed by western blotting. (E,F) HUVECs were pretreated with or without 5 mmol/L 3-MA followed by 10 ng/mL TNFa (E) or 10 ng/mL IL-1ß (F) for indicated time. VCAM-1 and ICAM-1 were analyzed by western blotting.

Fig 5: 3-MA inhibits late-phase I?Ba degradation. (A–D) HUVECs were pretreated with or without 5 mmol/L 3-MA for 30 min followed by 10 ng/mL TNFa (A,C) or 10 ng/mL IL-1ß (B,D) for indicated time. I?Ba, I?Bß and I?Be were analyzed by western blotting. (E,F) I?Ba blots of TNFa or IL-1ß control vs. 3-MA or MG-132 treatment (n = 3) at each time point were quantified by densitometry. (G) HUVECs were transfected with siRNA for 72 h followed by 10 ng/mL IL-1ß or 10 ng/mL TNFa for 6 h. I?Ba, I?Bß, I?Be and Atg5 were analyzed by western blotting.